The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Histology is the study of groups of specialised cells called tissues that are found in most multi-cellular plants and animals. Histological investigation includes study of tissue disease and regeneration and the reaction of tissue to injury or invading organisms. Because normal tissue has a characteristic appearance, histologic examination, such as by immunohistochemistry (IHC) and in situ hybridisation (ISH) is often utilised to identify diseased tissue and tissue morphology.
IHC and ISH both seek to detect a detectable entity in a sample by using specific binding agents capable of binding to the detectable entity. In IHC the specific binding agent generally comprises an antibody, and the detectable entity comprises a polypeptide, protein, or epitope comprised therein. ISH generally comprises detection of a target nucleic acid (such as DNA or RNA) in a sample, via the use of a specific labelled binding agent or probe such as a nucleic acid probe. Both IHC and ISH are routinely used to assist in the differential diagnosis of diseased and normal tissue such as in breast cancer diagnosis.
Evaluation of histological analysis based on visual estimation of the intensity of staining is often imprecise due to a variety of problems that may arise during the assay procedure. This analysis can be greatly improved by the use of appropriate control slides. In a clinical setting, the use of controls, such as positive and negative controls in IHC and ISH is essential.
Controls may be generated, for example, from a characterized archival tissue block or from cell lines embedded in a tissue support medium. In IHC, positive control testing is performed on sections of tissue known to contain the target antigen, processed using the same fixation, epitope retrieval and immunostaining protocols as the patient tissue. A separate tissue section may be used as a positive control, but test sections often contain normal elements that express the antigen of interest (internal controls).
A negative reagent control may be used to assess non-specific staining in patient tissue. A separate section of patient tissue is processed using the same reagent and epitope retrieval protocol as the patient test slide, except that the primary antibody is omitted, and replaced by any of the following: an unrelated antibody of the same isotype as the primary antibody (for monoclonal primary antibodies); an unrelated antibody from the same animal species as the primary antibody (for polyclonal primary antibodies); or the negative control reagent included in the staining kit. A separate negative reagent control should be run for each block of patient tissue being immunostained.
Negative tissue controls are tissues known not to express the antigen of interest, and as such should show no staining if the staining assay is functioning correctly. However, in both positive and negative controls, since the tissue used is non-standardized, it is never fully characterized and so there is always some doubt as to the control's usefulness.
IHC and ISH techniques require a series of treatment steps conducted on a tissue section mounted on a glass slide or other planar support to highlight by selective staining certain morphological indicators of disease states. Thus, for example in IHC, a sample is taken from an individual, fixed and exposed to antibodies against the antigen of interest. Further processing steps, for example, antigen retrieval, exposure to secondary antibodies (usually coupled to a suitable enzyme), washing, and to chromogenic enzyme substrates, etc may be necessary to reveal the pattern of antigen binding.
However, although it is possible with such kits to establish the reference levels of staining, there exists considerable difficulty in establishing a consistent quality of staining of the samples themselves. This arises from a variety of different factors, including non-homogeneous tissue material, the laborious and complex nature of the procedures, variability in reagent quality (including antibody/probe affinity and specificity), the technique of the operator sectioning the material and the subjective nature of the interpretation carried out by the practitioner. Furthermore, other sources of variability in sample staining include the conditions under which tissue samples are collected, processed and stored, variability in epitope retrieval procedures, and enzyme catalysed chromogen precipitation.
At present, the only method available for performing quality control on cut sections is staining by, for example, immunohistochemistry. Testing in this manner is destructive, as once control slides have been stained; they can no longer be supplied as an unstained product to customers. Further, whilst testing in this manner can indicate the quality of a particular section, it does not provide information as to the quality of all unstained sections, especially since the process of preparing numerous cut sections is considerably variable.
Variability in immunohistochemical staining intensity in control cell lines can be the consequence of an additional number of factors. These include any one of, or combination of, methods employed for cell culture, cell line processing such as length and type of fixation, cell line morphology, the percentage confluence at which cells were harvested, the techniques used to harvest cells (enzymatic or physical) and retrieval conditions used during the immunochemistry protocol.
With an increasing demand for control slides in histology, there is a need for a non-destructive method of manufacturing and identifying suitable control slides which can be reliably stained with minimal variability.